Sortase A (SrtA) is a membrane bound enzyme which attaches proteins covalently to the bacterial cell wall. The specific recognition motif on the SrtA substrate is LPXTG, whereby the enzyme cleaves between the amino acid residues threonine and glycine. The recognition motif on the peptidoglycan is a pentaglycine motif. It has been shown that a triglycine and even a diglycine motif on the N-terminus is sufficient to support the SrtA reaction (Clancy, K. W., et al., Peptide science 94 (2010) 385-396). The reaction proceeds through a thioester acyl-enzyme intermediate, which is resolved by the attack of an amine nucleophile from the oligoglycine, covalently linking peptidoglycan to a protein substrate and regenerating SrtA. SrtA can be used to covalently conjugate chemically synthetized peptides to recombinantly expressed proteins.
In WO 2010087994 methods for ligation and uses thereof are reported. Recombinant approaches to IgG-like bispecific antibodies are reported by Marvin, J. S., et al. (Acta Pharmacol. Sinica 26 (2005) 649-658). Levary, D. A., et al. (PLoS one, 6 (2011) e18342.1-e18342.6) report protein-protein fusion catalyzed by sortase A. In WO 2013/003555 the use of sortases to install click chemistry handles for protein ligation is reported.
Strijbis, K. et al (Traffic 13 (2012) 780-789) report protein ligation in living cells using Sortase. It has been stated by them that the Ca2+-dependent S. aureus sortase A is not functional intracellularly, but that the Ca2+-independent S. pyogenes Sortase A is functional in the cytosol and endoplasmic reticulum (ER) lumen of both Saccharomyces cerevisiae and mammalian HEK293T cells.
Levary, D. A., et al., report protein-protein fusion catalyzed by Sortase A (PLOS ONE 6 (2011)). Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by Sortase A-mediated protein ligation is reported by Madej, M. P., et al. (Biotechnol. Bioeng. 109 (2012) 1461-1470). Ta, H. T., et al., report enzymatic single-chain antibody tagging as a universal approach to targeted molecular imaging and cell homing in cardiovascular diseases (Cir. Res. 109 (2011) 365-373). Popp, M., et al., report making and breaking peptide bonds—protein engineering using sortase (Angew. Chem. Int. Ed. Eng. 50 (2011) 5024-5032). In WO 2010/087994 methods for ligation and uses thereof are reported. Engineered proteins with high affinity for DOTA chelates are reported in WO 2010/099536.
A truncated SrtA, that lacks the N-terminal membrane-anchoring motif, has been used for cell-surface protein labeling, covalent protein immobilization and incorporation of novel functionality into proteins. However, yields of SrtA-mediated ligation are always lower than 70%, if using equimolar amounts of substrate, because the reaction is reversible. Another drawback is the hydrolysis of the reaction intermediate which leads to a LPXT product which is not the intended one. This is especially problematic by long periods of incubation with SrtA. That although means that even small amounts of SrtA left in the final product can destroy it over time; this is a big issue for biologics where quality standards are very high.
Different efforts to block the revers reactions of Sortase have been reported. Yamamura, Y., et al. (Chem. Commun. 47 (2011) 4742-4744) reported enhancement of sortase A-mediated protein ligation by inducing a beta-hairpin structure around the ligation site by introducing a β-hairpin around the recognition site of the substrate.
Sorting of LPXTG peptides by archetypal sortase A, role of invariant substrate residues in modulating the enzyme dynamics and conformational signature of a productive substrate was reported by Biswas, T., et al. (Biochem. 53 (2014) 2515-2524).
Li, Y. M., et al. report irreversible site-specific hydrazinolysis of proteins by use of Sortase (Angew. Chem. Int. Ed. Engl. 53 (2014) 2198-2202).
In WO 2014/001324 a method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof is reported. Marraffini, L. A., et al. (J. Biol. Chem. 279 (2004) 37763-37770) report for anchoring of surface proteins to the cell wall of staphylococcus aureus a conserved arginine residue is required for efficient catalysis of Sortase A.
However, all these approaches have the drawback, that they produce or employ an artificial motive or structure, which may result later on in problems in vivo, like immunogenicity.